Three investigations were conducted in order to investigate the effect of dietary probiotics on tilapia (Oreochromis niloticus) growth performance, intestinal morphology, intestinal microbiology and immunity. The first experiment demonstrated that Bacillus subtilis, Lactobacillus reuteri and Pediococcus acidilactici supplemented individually and as a mixed probiotic (in addition to Enterococcus faecium; AquaStar® Growout) were capable of modulating intestinal microbial populations as determined by culture dependent methods and DGGE. Furthermore, high-throughput sequencing reported that >99% of 16S rRNA reads in the mixed probiotic group belonged to the probiotic genera, predominantly assigned to Enterococcus (52.50%) and Bacillus (45.94%). Tilapia in the mixed probiotic group displayed significantly higher intraepithelial leucocyte (IEL) populations in the mid intestine when compared to the control and L. reuteri treatment. The mixed probiotic also improved microvilli density and had a higher absorptive surface area when compared to the control. In the second trial, after six weeks of supplementing tilapia diets with AquaStar® Growout at 3g kg-1, fish demonstrated significantly higher final weight, weight gain and SGR when compared to that of the control (void of probiotic) treatment or an initial probiotic feed (lasting two weeks) followed by control feeding. Probiotic supplementation at 3g kg-1 also caused an increase in the abundance of intestinal IELs and goblet cells and an up-regulation in the gene expression of intestinal caspase-3, PCNA and HSP70 and immunity genes TLR2, TNFα, IL-1β, TGFβ and IL-10 when compared with the expression of control replicates. These changes were not observed when supplementing tilapia diets with a lower dose (1.5g kg-1), nor when supplementing the probiotic in either a pulsed manner or as an initial feed (two weeks) followed by control feeding. Trial three revealed that the probiotic had a more discrete effect on the intestinal allochthonous microbiota as 16S rRNA reads assigned to probiotic genera only accounted for 5-10% of total reads. Nevertheless, the supplementation of dietary AquaStar® Growout at 3g kg-1 improved the localised immune response in tilapia, through the regulation of immunity genes TLR2, MYD88, NFκB, TNFα, IL-1β, TGFβ and IL-10, larger populations of goblet cells and a higher recruitment of IELs. Furthermore, the probiotic also improved the systemic immune response through the regulation of immunity genes (mentioned above) in the head kidney and significantly higher circulating leucocyte levels in whole blood. The extent of these changes were dependent on the probiotic treatment (i.e. continuously supplemented in feed or alternating weekly between probiotic at 3 g kg-1 and control feeding), the duration of feeding and the parameter investigated. This research demonstrates that B. subtilis, L. reuteri, P. acidilactici and AquaStar® Growout can modulate the intestinal microbiota. In addition, AquaStar® Growout can improve intestinal morphology, growth performance and modulate both the localised and systemic immune responses of tilapia when supplemented through the feed at the appropriate dosage and feeding regime.

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